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Since their discovery in insects, pheromones are considered as ubiquitous and stereotyped chemical messengers acting in intraspecific animal communication. Here we studied the effect of pheromones in a different context as we investigated their capacity to induce persistent modulations of associative learning and memory. We used honey bees, Apis mellifera, and combined olfactory conditioning and pheromone preexposure with disruption of neural activity and two-photon imaging of olfactory brain circuits, to characterize the effect of pheromones on olfactory learning and memory. Geraniol, an attractive pheromone component, and 2-heptanone, an aversive pheromone, improved and impaired, respectively, olfactory learning and memory via a durable modulation of appetitive motivation, which left odor processing unaffected. Consistently, interfering with aminergic circuits mediating appetitive motivation rescued or diminished the cognitive effects induced by pheromone components. We thus show that these chemical messengers act as important modulators of motivational processes and influence thereby animal cognition.

As honey bee populations worldwide are declining there is an urgent need for a deeper understanding of stress reactivity in these important insects. Our data indicate that stress responses in bees (Apis mellifera L.) may be mediated by neuropeptides identified, on the basis of sequence similarities, as allatostatins (ASTA, ASTC and ASTCC). Effects of allatostatin injection are compared with stress-related changes in learning performance induced by the honeybee alarm pheromone, isopentylacetate (IPA). We find that bees can exhibit two markedly different responses to IPA, with opposing effects on learning behaviour and memory generalisation, and that strikingly similar responses can be elicited by allatostatins, in particular ASTCC. These findings lend support to the hypothesis that allatostatins mediate stress reactivity in honey bees and suggest responses to stress in these insects are state dependent.

Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee Apis mellifera, olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM) are formed upon multiple-trial conditioning: an early phase (e-LTM) which depends on translation from already available mRNA, and a late phase (l-LTM) which requires de novo transcription and translation. Here we combined olfactory PER conditioning and neuropharmacological inhibition and studied the involvement of the NO-cGMP pathway, and of specific molecules, such as cyclic nucleotide-gated channels (CNG), calmodulin (CaM), adenylyl cyclase (AC), and Ca(2+)/calmodulin-dependent protein kinase (CaMKII), in the formation of olfactory LTM in bees. We show that in addition to NO-cGMP and cAMP-PKA, CNG channels, CaM, AC, and CaMKII also participate in the formation of a l-LTM (72-h post-conditioning) that is specific for the learned odor. Importantly, the same molecules are dispensable for olfactory learning and for the formation of both MTM (in the minute and hour range) and e-LTM (24-h post-conditioning), thus suggesting that the signaling pathways leading to l-LTM or e-LTM involve different molecular actors.

The insect mushroom bodies (MBs) are paired brain centers which, like the mammalian hippocampus, have a prominent function in learning and memory. Despite convergent evidence for their crucial role in the formation and storage of associative memories, little is known about the mechanisms underlying such storage. In mammals and other species, the consolidation of stable memories is accompanied by structural plasticity involving variations in synapse number and/or size. Here, we address the question of whether the formation of olfactory long-term memory (LTM) could be associated with changes in the synaptic architecture of the MB networks. For this, we took advantage of the modular architecture of the honeybee MB neuropil, where synaptic contacts between olfactory input and MB neurons are segregated into discrete units (microglomeruli) which can be easily visualized and counted. We show that the density in microglomeruli increases as a specific olfactory LTM is formed, while the volume of the neuropil remains constant. Such variation is reproducible and is clearly correlated with memory consolidation, as it requires gene transcription. Thus stable structural synaptic rearrangements, including the growth of new synapses, seem to be a common property of insect and mammalian brain networks involved in the storage of stable memory traces.

The storage of stable memories is generally considered to rely on changes in the functional properties and/or the synaptic connectivity of neural networks. However, these changes are not easily tractable given the complexity of the learning procedures and brain circuits studied. Such a search can be narrowed down by studying memories of specific stimuli in a given sensory modality and by working on networks with a modular and relatively simple organization. We have therefore focused on associative memories of individual odors and the possible related changes in the honeybee primary olfactory center, the antennal lobe (AL). As this brain structure is organized in well-identified morpho-functional units, the glomeruli, we looked for evidence of structural and functional plasticity in these units in relation with the bees' ability to store long-term memories (LTMs) of specific odors. Restrained bees were trained to form an odor-specific LTM in an appetitive Pavlovian conditioning protocol. The stability and specificity of this memory was tested behaviorally 3 d after conditioning. At that time, we performed both a structural and a functional analysis on a subset of 17 identified glomeruli by measuring glomerular volume under confocal microscopy, and odor-evoked activity, using in vivo calcium imaging. We show that long-term olfactory memory for a given odor is associated with volume increases in a subset of glomeruli. Independent of these structural changes, odor-evoked activity was not modified. Lastly, we show that structural glomerular plasticity can be predicted based on a putative model of interglomerular connections.

It is generally accepted that, after learning, memories stabilize over time and integrate into long-term memory (LTM) through the process of consolidation, which depends on de novo protein synthesis. Besides, studies on several species have shown that reactivation of already stabilized LTM can either make this memory labile and then restabilize it (a process called reconsolidation) or inhibit it (extinction). However, the identity of both processes and their interactions with consolidation are still under debate. Regarding memory stabilization, Drosophila offers a striking exception since, in this species, LTM is not the sole stable form of memory. Under specific learning conditions, anesthesia-resistant memory (ARM) can be formed through processes yet unknown but that are resistant to cycloheximide, a classical protein synthesis inhibitor that impairs LTM. Here, we took advantage of this dichotomy to ask whether both ARM and LTM could be extinguished and/or reconsolidated. We also studied whether two forms of memory extinction and reconsolidation exist in flies, as for memory stabilization. We show that either reconsolidation or extinction can be induced after olfactory conditioning in Drosophila, depending on the number of reactivations as in other species. Furthermore, regarding the effect of cycloheximide, the ARM/LTM dichotomy for stabilization does not apply to extinction and reconsolidation. Blocking protein synthesis interfered with both processes regardless of whether initial stabilization was sensitive (LTM) or not (ARM) to cycloheximide. These results thus show that Drosophila is a useful model to tackle these questions and that reconsolidation is not necessarily a mere repetition of consolidation.